15N Nuclear Magnetic Resonance Studies of Acid-Base Properties of Pyridoxal-5'-Phosphate Aldimines in Aqueous Solution

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• 作者：Shasad Sharif ; Monique Chan Huot ; Peter M. Tolstoy ; Michael D. Toney ; K. Hanna M. Jonsson ; Hans-Heinrich Limbach
• 刊名：Journal of Physical Chemistry B
• 出版年：2007
• 出版时间：April 19, 2007
• 年：2007
• 卷：111
• 期：15
• 页码：3869 - 3876
• 全文大小：183K
• 年卷期：v.111,no.15(April 19, 2007)
• ISSN：1520-5207

文摘

By use of ¹⁵N NMR spectroscopy, we have measured the pK_a values of the aldimines ¹⁵N-(pyridoxyl-5'-phosphate-idine)-methylamine (2a), N-(pyridoxyl-5'-phosphate-¹⁵N-idine)-methylamine (2b), and ¹⁵N-(pyridoxyl-idine)-methylamine (3). These aldimines model the cofactor pyridoxal-5'-phosphate (PLP, 1) in a varietyof PLP-dependent enzymes. The acid-base properties of the aldimines differ substantially from those of thefree cofactor in the aldehyde form 1a or in the hydrated form 1b, which were also investigated using ¹⁵NNMR for comparison. All compounds contain three protonation sites, the pyridine ring, the phenol group,and the side chain phosphate (1, 2) or hydroxyl group (3). In agreement with the literature, 1a exhibits oneof several pK_as at 2.9 and 1b at 4.2. The ¹⁵N chemical shifts indicate that the corresponding deprotonationoccurs partially in the pyridine and partially in the phenolic site, which compete for the remaining proton.The equilibrium constant of this ring-phenolate tautomerism was measured to be 0.40 for 1a and 0.06 for 1b.The tautomerism is essentially unaltered above pH 6.1, where the phosphate group is deprotonated to thedianion. This means that the pyridine ring is more basic than the phenolate group. Pyridine nitrogendeprotonation occurs at 8.2 for 1a and at 8.7 for 1b. By contrast, above pH 4 the phosphate site of 2 isdeprotonated, while the pyridine ring pK_a is 5.8. The Schiff base nitrogen does not deprotonate below pH11.4. When the phosphate group is removed, the pK_a of the Schiff base nitrogen decreases to 10.5. Thephenol site cannot compete for the proton of the Schiff base nitrogen and is present in the entire pH rangeas a phenolate, preferentially hydrogen bonded to the solvent. The intrinsic ¹⁵N chemical shifts provideinformation about the hydrogen bond structures of the protonated and unprotonated species involved. Evidenceis presented that the intramolecular OHN hydrogen bond of PLP aldimines is broken in aqueous solution.The coupling between the inter- and intramolecular OHN hydrogen bonds is also lost in this environment.The pyridine ring of the PLP aldimines is not protonated in aqueous solution near neutral pH. The basicityof the aldimine nitrogens would be even lower without the doubly negatively charged phosphate group.Protonation of both the Schiff base and pyridine nitrogens has been discussed as a prerequisite for catalyticactivity, and the implications of the present findings for PLP catalysis are discussed.